Two aspects of this project: summarized below in abstract form:1. The cis-acting elements that control the highly regulated expression of the cystic fibrosis transmembrane conductance regulator gene (CFTR) remain ill defined. The construction of a yeast artificial chromosome (YAC)-based reporter gene that encompasses hundreds of kilobases of CFTR flanking DNA should aid in elucidating these DNA elements. Therefore, a 350 kb YAC containing a luciferase reporter gene (luc) driven by ~335 kb of CFTR 5-flanking DNA was constructed and transferred into human HT-29 cells. Stable, clonal cell lines containing the YAC were assessed for luciferase activity at baseline and response to forskolin and the phorbol 12-myristate 13-acetate (PMA). The baseline luciferase activity fell within a wide range among the clones (1-5094 units/mg protein). However, irrespective of the baseline value, an increase in activity was observed in response to forskolin. In contrast to published reports an increase in luciferase activity was also observed in response to PMA. However, the endogenous HT-29 CFTR responded with an increase in expression in response to forskolin and a decrease in response to PMA. In addition, a second YAC (y5lucI) was similarly constructed but with the addition of a putative enhancer element from CFTR intron 1. Stable Chinese hamster ovary (CHO-K1) cell clones were derived using each YAC and used to assess the role that luc copy number and the presence of intron 1 played in the luc expression. The CHO-K1 lines demonstrated a wide range of luciferase activity irrespective of luc copy number. On average, luciferase activity was higher in the clones containing y5lucI. These data are consistent with a positive role for intron 1 in regulating CFTR expression and suggest the feasibility of using this system for further assessing cis-acting elements that control CFTR expression.2. The goals of this part of the study are (1) to develop a physiologically-relevant CFTR transfer vector and (2) to determine whether introduction of the full-length CFTR gene into CF airway epithelial cell lines rescues the CF phenotype. Our 610 kb yeast artificial chromosome (YAC) which includes the 250 kb CFTR gene, 330 kb of 5 and 30 kb of 3 flanking sequence, was retrofitted with a neomycin resistance gene (neor), and modified to include two unique restriction sites in the 3 untranslated region (UTR) of exon 24 of CFTR which allow DNA and mRNA derived specifically from the YAC to be uniquely identified. This YAC generates full length CFTR message and functional protein in Chinese hamster ovary cells. In the present study, the YAC DNA was transfected into two CF airway epithelial cell lines: CFT1 (Yankaskas et al. AJP-Cell 264:C1219-1230, 1993) and IB3-1 (Zeitlin et al., Am J. Respir. Cell Mol. Biol 4:313-9, 1991). Twelve neoor clones (5 from CFT1 and 7 from IB3-1) were derived (Compton et al., NAR 27:1762-5, 1999). Nested RT-PCR and restriction digestion of the 3-UTR indicated that all 12 clones expressed CFTR mRNA from the YAC. As expected for cells which express CFTR at physiological levels, two rounds of PCR were required to detect the presence of endogenous and YAC-derived CFTR transcripts in the clones, as well as in a normal airway epithelial cell line, HBE (Yankaskas et al. AJP-Cell 264:C1219-1230, 1993). The 36Cl efflux assay was used to screen the clones for a cAMP-dependent increase in chloride permeability. While a robust response was found in the Calu-3 cell line, known to express CFTR at high levels, no response was observed in HBE or any of the 12 CFT YAC derived clones (n=6 each cell line). Based on these results we hypothesized that this assay might not be sensitive enough to detect the level of CFTR function in these cells. Thus, we pursued other maneuvers and functional assays to distinguish the YAC clones from the parental cell lines. Exposure of one of the CFT YAC clones, Y-7, to serum for 36 hours prior to efflux, resulted in a small cAMP-dependent increase in chloride permeability. In addition, preliminary evidence indicates that basal and hypotonically-stimulated ATP release into the apical medium was significantly augmented in Y-7 and one additional CFT YAC clone compared to a neor clone which does not contain the CFTR YAC. These data support the hypothesis that CF epithelia exhibit a deficit in extracellular ATP signaling. FISH and Southern blot analyses indicate that CFTR has been amplified in at least 3 of the CFT1 clones. Future studies are aimed at (1) screening the YAC clones using functional assays that are able to distinguish a CF from a non-CF phenotype in cells which express physiologic levels of CFTR, and (2) determining why CFTR amplification does not correlate with increased cAMP-dependent chloride permeability.